Proper regulation of NF-?B transcription factors is required to mount immune responses to pathogens and for the normal homeostasis of the immune system. Conversely, excessive NF-?B-dependent inflammation can help to drive the development of autoimmune disease and the formation of tumors. In this light, it is important to know how NF-?B family transcription factors are controlled by signaling through the different types of receptors that impinge on this pathway. During T cell activation by antigen, TCR and CD28, and numerous signaling proteins, are reorganized into distinct structures while undergoing well-described post-translational modifications, such as phosphorylation and ubiquitination. Although the IKK complex accumulates in the TCR-rich domain at the center of the immune synapse, receptor signals are initiated by TCR 'microclusters' in the periphery of the contact. We have found that the IKK complex is rapidly recruited to signaling microclusters containing the TCR and Zap70, but not SLP-76. Our imaging studies have also revealed additional pools of IKK? that may be involved in delivery of IKK? to TCR microclusters and/or its down-regulation. However, a number of important questions remain to be answered regarding the sub-cellular regulation of IKK? and its role in T cell activation. Carrying out such studies is important, if we are to eventually exploit this knowledge to manipulate TCR-mediated NF-?B activation for therapeutic purposes. We hypothesize that TCR/CD28 activation of the classical NF-?B pathway requires complex sub-cellular regulation of the adaptor protein IKK?/NEMO. Thus, in Aim 1, we will determine the role of Zap70 and Lck in recruitment of the IKK complex to TCR microclusters. In Aim 2, we will define the role of Carma1 and associated proteins in spatial regulation of the IKK complex. Finally, in Aim 3 we will define the sub-cellular compartment(s) containing total and active IKK complexes